The present invention relates to an apparatus, media, and method for DNA evaluation.
Genotyping is the discipline of identifying an individual""s genome in relation to disease specific alleles and/or mutations that occur as an effect of parental linkage. The rapid purification of human genomic DNA is an essential part of a genotyping process; the genomic DNA of an individual being the structural unit for the entire DNA sequence of every allele expressed.
Human genomic DNA cannot be directly sequenced. In order to carry out sequence analysis on regions of the chromosomes that may contain portions of mutation or disease specific sequences, selected portions are amplified via PCR and the amplified products are sequenced. The selected portions of the chromosomes that are amplified are dictated by the specific sequence of the primers used in the PCR amplification. The primer sets that are used in genotyping studies are commercially available and are representative for the chromosome under examination. If linkage studies identify that a disease bearing sequence is on a particular chromosome, then many primer sets will be utilized across that chromosome in order to obtain genetic material for sequencing. The resultant PCR products may well represent the entire chromosome under examination. Due to the large length of chromosomes, many PCR reactions are carried out on the genomic DNA template from a single patient.
Human genomic DNA is purified by a variety of methods (Molecular Cloning, Sambrook et al. (1989)). Consequently, many commercial kit manufacturers provide products for such techniques, for example: AmpReady(trademark) (Promega, Madison, Wis.), DNeasy(trademark) (Qiagen, Valencia, Calif.), and Split Second(trademark) (Roche Molecular Biochemicals, Indianapolis, Ind.). These products rely on the use of specialized matrices or buffer systems for the rapid isolation of the genomic DNA molecule.
More recently, microporous filter-based techniques have surfaced as tools for the purification of genomic DNA as well as a whole multitude of nucleic acids. The advantage of filter-based matrices are that they can be fashioned into many formats that include tubes, spin tubes, sheets, and microwell plates. Microporous filter membranes as purification support matrices have other advantages within the art. They provide a compact, easy to manipulate system allowing for the capture of the desired molecule and the removal of unwanted components in a fluid phase at higher throughput and faster processing times than possible with column chromatography. This is due to the fast diffusion rates possible on filter membranes.
Nucleic acid molecules have been captured on filter membranes, generally either through simple adsorption or through a chemical reaction between complementary reactive groups present on the filter membrane or on a filter-bound ligand resulting in the formation of a covalent bond between the ligand and the desired nucleic acid.
Porous filter membrane materials used for non-covalent nucleic acid immobilization have included materials such as nylon, nitrocellulose, hydrophobic polyvinylidinefluoride (PVDF), and glass microfiber. A number of methods and reagents have also been developed to also allow the direct coupling of nucleic acids onto solid supports, such as oligonucleotides and primers (eg. J. M. Coull et al., Tetrahedron Lett. Vol. 27, page 3991; B. A. Conolly, Nucleic Acids Res., vol. 15, page 3131, 1987; B. A. Conolly and P. Rider, Nucleic Acids Res., vol. 12, page 4485, 1985; Yang et al P.N.A.S. Vol.95: 5462-5467). UV cross-linking of DNA (Church et al., PNAS, vol. 81, page 1991, 1984), The Generation Capture Column Kit (Gentra Systems, Minneapolis, Minn.) and RNA (Khandjian, et al., Anal. Biochem, Vol. 159, pages 227, 1986) to nylon membranes have also been reported.
Many chemical methods have been utilized for the immobilization of molecules such as nucleic acids on filter membranes. For example, activated paper (TransBind.TM, Schleicher and Schuell Ltd., Keene, N.H.) carbodimidazole-activated hydrogel-coated PVDF membrane (Immobilin-IAV.TM, Millipore Corp., Bedford, Mass.), MAP paper (Amersham, Littlechalfont Bucks, Wis.), activated nylon (BioDyne. TM, Pall Corp., (Glen Cove, N.Y.), DVS- and cyanogen bromide-activated nitrocellulose. Membranes bound with specific ligands are also known such as the SAM2TM Biotin Capture Membrane (Promega) which binds biotinylated molecules based on their affinity to streptavidin or MAC affinity membrane system (protein A/G) (Amicon, Bedford, Mass.). Some of the disadvantages of covalent attachment of biomolecules onto activated membranes are:
a) Molecule immobilization is often slow requiring 20-180 minutes for reaction completion.
b) High ligand and biomolecule concentration is needed for fast immobilization.
c) Constant agitation is needed during the immobilization process that may result in biomolecule denaturation and deactivation.
d) Once the immobilization process is complete, often a blocking (capping) step is required to remove residual covalent binding capacity.
e) Covalently bound molecules can not be retrieved from the filter membrane.
There is a need in various specific areas, such as forensics, for a nucleic acid immobilization media and procedure that exhibits the high specificity of covalent immobilization onto the filter membrane without the use of harsh chemical reactions and long incubation times, which can also be used at crime scenes, with blood sample archiving and other related uses. In particular there is a need for the capture and separation of nucleic acids from a mixture in a fluid phase onto a filter membrane matrix in forensics. Of special interest is the ability to store or archive the bound nucleic acids on the filter membrane matrix for such uses.
More recently, glass microfiber, has been shown to specifically bind nucleic acids from a variety of nucleic acid containing sources very effectively (for example see: Itoh et al (1997) Simple and rapid preparation of plasmid template by filtration method using microtiter filter plates. NAR, vol. 25, No. 6: 1315-1316; Andersson, B. et al (1996) Method for 96-well M13 DNA template preparations for large-scale sequencing. BioTechniques vol. 20: 1022-1027). Under the correct salt and buffering conditions, nucleic acids will bind to glass or silica with high specificity.
Based on U.S. Pat. Nos. 5,496,562, 5,756,126, and 5,807,527, it has been demonstrated that nucleic acids or genetic material can be immobilized to a cellulosic-based dry solid support or filter (FTA filter). The solid support described is conditioned with a chemical composition that is capable of carrying out several functions: (i) lyse intact cellular material upon contact, releasing genetic material, (ii) enable and allow for the conditions that facilitate genetic material immobilization to the solid support (probably by a combination of mechanical and chaotrophic), (iii) maintain the immobilized genetic material in a stable state without damage due to degradation, endonuclease activity, UV interference, and microbial attack, and (iv) maintain the genetic material as a support-bound molecule that is not removed from the solid support during any down stream processing (as demonstrated by Del Rio et al (1995) BioTechniques. Vol. 20: 970-974).
The usefulness of the so called FTA cellulosic filter material described in U.S. Pat. Nos. 5,496,562, 5,756,126, and 5,807,527 has been illustrated for several nucleic acid techniques such as bacterial ribotyping (Rogers, C and Burgoyne, L (1997) Anal. Biochem. Vol. 247: 223-227), detection of single base differences in viral and human DNA (Ibrahim et al (1998) Anal. Chem. Vol. 70: 2013-2017), DNA databasing (Ledray et al (1997) J. Emergency Nursing. Vol.23, No. 2: 156-158), automated processing for STR electrophoresis (Belgrader, B and Marino, M (1996) L.R.A. vol.9: 3-7, Belgrader et al (1995) BioTechniques. Vol. 19, No. 3: 427-432), and oligonucleotide ligation assay for diagnostics (Baron et al (1996) Nature Biotech. Vol 14: 1279-1282).
It has been shown that nucleic acid or genetic material applied to, and immobilized to, FTA filters cannot be simply removed, or eluted from the solid support once bound (Del Rio et al (1995) BioTechniques. Vol. 20: 970-974). This is a major disadvantage for applications where several downstream processes are required from the same sample, such a STR profiling and genotyping.
Currently, cellular material is applied to FTA filter media, and generally the cellular material, once applied forms a spot on the FTA filter. From this spot, small punches can be taken; each small punch will have immobilized to it enough nucleic acid or genetic material to facilitate a single downstream process such as a PCR reaction. As the two primers administered to a PCR reaction are presented in solution, it is of no consequence that the cellular nucleic acid template is immobilized to the filter. All amplicon will be formed in solution. Amplicon can then be readily removed from the reaction by aspirating the liquid phase away from the FTA solid filter punch. Therefore, for multiple processing from a single sample, many punches have to be taken. Multiple punching is very time consuming, and as yet, has not lent itself to simplified automation.
It is much more desirable to provide nucleic acid as a soluble fraction from which aliquots can be readily dispensed to as many reactions as required. Automated liquid handling of this type is a fundamental technique within the pharmaceutical and other industries (for example see: Armstrong et al (1998) J. Biomolecular Screening. Vol. 3, No. 4: 271-275).
It is desirable to adapt the present technology and modify it for specific use in the forensic art Additionally, it would be advantageous to be able to rapidly qualify and quantify nucleic acid on media, either in correlation with such uses, or independent thereof.
According to the present invention there is provided a substrate for lysing cells and purifying nucleic acid having a matrix and a coating and an integrity maintainer for maintaining the purified nucleic acid. Also provided is a method of purifying nucleic acid by applying a nucleic acid sample to a substrate, the substrate physically capturing the nucleic acid, bonding the nucleic acid to the substrate and generating a signal when the nucleic acid bonds to the substrate indicating the presence of the nucleic acid. A kit for purifying nucleic acid containing a coated matrix and an integrity maintenance provider for preserving the matrix and purifying nucleic acid is also provided.